Method for identifying lineage-related antibodies

ABSTRACT

In certain embodiments, the method may comprise: a) obtaining the antibody sequences from a population of B cells; b) grouping the antibody sequences to provide a plurality of groups of lineage-related antibodies; c) testing a single antibody from each of the groups in a bioassay and, after the first antibody has been identified, d) testing further antibodies that are in the same group as the first antibody in a second bioassay. In another embodiment, the method may comprise: a) testing a plurality of antibodies obtained from a first portion of an antibody producing organ of an animal; b) obtaining the sequence of a first identified antibody; c) obtaining from a second portion of said antibody producing organ the sequences of further antibodies that are related by lineage to said first antibody; and, c) testing the further antibodies in a second bioassay.

CROSS-REFERENCING

This patent application is a continuation of U.S. patent applicationSer. No. 13/552,517, filed Jul. 18, 2012, now U.S. Pat. No. 8,617,830,which is a continuation of U.S. patent application Ser. No. 12/878,925,filed on Sep. 9, 2010, now U.S. Pat. No. 8,293,483, which claims thebenefit of U.S. provisional application Ser. No. 61/241,714, filed onSep. 11, 2009, which application is incorporated by reference herein inits entirety.

INTRODUCTION

Antibodies are proteins that bind a specific antigen. Generally,antibodies are specific for their targets, have the ability to mediateimmune effector mechanisms, and have a long half-life in serum. Suchproperties make antibodies powerful therapeutics. Monoclonal antibodiesare used therapeutically for the treatment of a variety of conditionsincluding cancer, inflammation, and other diseases. There are currentlyover two dozen therapeutic antibody products on the market and hundredsin development.

There is a constant need for new antibodies and methods for making thesame.

SUMMARY

In certain embodiments, the method may comprise: a) obtaining theantibody heavy chain sequences and the antibody light chain sequencesfrom a population of B cells of an animal, wherein the population of Bcells is enriched for B cells that produce antibodies that specificallybind to a target antigen; b) grouping the heavy and light chainsequences on the basis of sequence similarity to provide a plurality ofgroups of antibodies that are related by lineage; c) testing a singleantibody from each of the groups in a first bioassay to identify a firstantibody that has a biological activity; and, after the first antibodyhas been identified, d) testing further antibodies that are in the samegroup as the first antibody in a second bioassay, thereby identifying asecond antibody that has the biological activity.

In other embodiment, the method may comprise: a) testing a plurality ofantibodies obtained from a first portion of an antibody producing organof an animal in a first bioassay to identify a first antibody that has abiological activity; b) obtaining the sequence of the first antibody; c)obtaining from a second portion of said antibody producing organ theheavy and light chain amino acid sequences of further antibodies thatare related by lineage to said first antibody by PCR, using probes aredesigned using the sequence of the first antibody; and, c) testing aplurality of the further antibodies in a second bioassay to identify asecond antibody that has said biological activity.

In certain embodiments, the method provides a means by which significantportion of the entire antibody repertoire of an animal can be screenedto identify an antibody with desirable properties. In certainembodiments the method involves first identifying a single antibody withdesirable properties, and then screening other antibodies in samelineage group (i.e., a clonally related group of antibodies) as theidentified antibody, to identify other antibodies that may have evenmore desirable properties relative to the identified antibody. As such,the method provides an efficient way to screen for and identify new,biologically active antibodies. After identification, the secondantibody may be tested in further assays, and, if it is suitable for useas a therapy, may be humanized, for example.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B schematically illustrate two embodiments.

FIG. 2 shows the amino acid sequences of selected KDR-bindingantibodies. Page 1 of FIG. 2 shows amino acid sequences of the heavychains. Page 2 of FIG. 2 shows amino acid sequences of the correspondinglight chains. The amino acid sequences shown in FIG. 2 are of antibodiesthat specifically bind to KDR and block VEGF activity. From top tobottom, FIG. 2 (page 1 of 2) SEQ ID NOS: 1-47 and FIG. 2 (page 2 of 2)SEQ ID NOS: 48-94.

FIG. 3 shows the amino acid sequence of 20 exemplary VH3 regions ofunrelated rabbit antibodies. From top to bottom SEQ ID NOS: 95-114.

FIGS. 4A-4H show exemplary methods by which related antibodies can beamplified.

DEFINITIONS

Before the present subject invention is described further, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anantibody” includes a plurality of such antibodies and reference to “aframework region” includes reference to one or more framework regionsand equivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

The term “nucleic acid” encompasses DNA, RNA, single stranded or doublestranded and chemical modifications thereof. The terms “nucleic acid”and “polynucleotide” are used interchangeably herein.

The term “expression”, as used herein, refers to the process by which apolypeptide is produced based on the nucleic acid sequence of a gene.The process includes both transcription and translation.

The term “expression cassette” refers to a nucleic acid constructcapable of directing the expression of a gene/coding sequence ofinterest, which is operably linked to a promoter of the expressioncassette. Such cassettes can be a linear nucleic acid or can be presentin a “vector”, “vector construct”, “expression vector”, or “genetransfer vector”, in order to transfer the expression cassette intotarget cells. Thus, the term includes cloning and expression vehicles,as well as viral vectors.

The term “operably linked” refers to an arrangement of elements whereinthe components so described are configured so as to perform their usualfunction. Thus, a given signal peptide that is operably linked to apolypeptide directs the secretion of the polypeptide from a cell. In thecase of a promoter, a promoter that is operably linked to a codingsequence will direct the expression of the coding sequence. The promoteror other control elements need not be contiguous with the codingsequence, so long as they function to direct the expression thereof. Forexample, intervening untranslated yet transcribed sequences can bepresent between the promoter sequence and the coding sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

The term “plurality” refers to more than 1, for example more than 2,more than about 5, more than about 10, more than about 20, more thanabout 50, more than about 100, more than about 200, more than about 500,more than about 1000, more than about 2000, more than about 5000, morethan about 10,000, more than about 20,000, more than about 50,000, morethan about 100,000, usually no more than about 200,000. A “population”contains a plurality of items.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, or ‘transformation”, or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell wherein the nucleicacid sequence may be present in the cell transiently or may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid, or mitochondrial DNA), converted into an autonomous replicon.

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. These terms are well understood by those in the field, and referto a protein consisting of one or more polypeptides that specificallybinds an antigen. One form of antibody constitutes the basic structuralunit of an antibody. This form is a tetramer and consists of twoidentical pairs of antibody chains, each pair having one light and oneheavy chain. In each pair, the light and heavy chain variable regionsare together responsible for binding to an antigen, and the constantregions are responsible for the antibody effector functions.

The recognized immunoglobulin polypeptides include the kappa and lambdalight chains and the alpha, gamma (IgG₁, IgG₂, IgG₃, IgG₄), delta,epsilon and mu heavy chains or equivalents in other species. Full-lengthimmunoglobulin “light chains” (of about 25 kDa or about 214 amino acids)comprise a variable region of about 110 amino acids at the NH₂-terminusand a kappa or lambda constant region at the COOH-terminus. Full-lengthimmunoglobulin “heavy chains” (of about 50 kDa or about 446 aminoacids), similarly comprise a variable region (of about 116 amino acids)and one of the aforementioned heavy chain constant regions, e.g., gamma(of about 330 amino acids).

The terms “antibodies” and “immunoglobulin” include antibodies orimmunoglobulins of any isotype, fragments of antibodies which retainspecific binding to antigen, including, but not limited to, Fab, Fv,scFv, and Fd fragments, chimeric antibodies, humanized antibodies,single-chain antibodies, and fusion proteins comprising anantigen-binding portion of an antibody and a non-antibody protein. Theantibodies may be detectably labeled, e.g., with a radioisotope, anenzyme which generates a detectable product, a fluorescent protein, andthe like. The antibodies may be further conjugated to other moieties,such as members of specific binding pairs, e.g., biotin (member ofbiotin-avidin specific binding pair), and the like. The antibodies mayalso be bound to a solid support, including, but not limited to,polystyrene plates or beads, and the like. Also encompassed by the termare Fab′, Fv, F(ab′)₂, and or other antibody fragments that retainspecific binding to antigen, and monoclonal antibodies.

Antibodies may exist in a variety of other forms including, for example,Fv, Fab, and (Fab′)₂, as well as bi-functional (i.e. bi-specific) hybridantibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987))and in single chains (e.g., Huston et al., Proc. Natl. Acad. Sci.U.S.A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426(1988), which are incorporated herein by reference). (See, generally,Hood et al., “Immunology”, Benjamin, N.Y., 2nd ed., 1984, andHunkapiller and Hood, Nature, 323, 15-16, 1986).

An immunoglobulin light or heavy chain variable region consists of aframework region (FR) interrupted by three hypervariable regions, alsocalled “complementarity determining regions” or “CDRs”. The extent ofthe framework region and CDRs have been precisely defined (see,“Sequences of Proteins of Immunological Interest,” E. Kabat et al., U.S.Department of Health and Human Services, 1991). The sequences of theframework regions of different light or heavy chains are relativelyconserved within a species. The framework region of an antibody, that isthe combined framework regions of the constituent light and heavychains, serves to position and align the CDRs. The CDRs are primarilyresponsible for binding to an epitope of an antigen.

The term “chimeric antibodies” refer to antibodies whose light and heavychain genes have been constructed, typically by genetic engineering,from antibody variable and constant region genes belonging to differentspecies. For example, the variable segments of the genes from a mousemonoclonal antibody may be joined to human constant segments, such asgamma 1 and gamma 3. An example of a therapeutic chimeric antibody is ahybrid protein composed of the variable or antigen-binding domain from arabbit antibody and the constant or effector domain from a humanantibody, although other mammalian species may be used.

The term “humanized antibody” or “humanized immunoglobulin” refers to anon-human (e.g., mouse or rabbit) antibody containing one or more aminoacids (in a framework region, a constant region or a CDR, for example)that have been substituted with a correspondingly positioned amino acidfrom a human antibody. In general, humanized antibodies produce areduced immune response in a human host, as compared to a non-humanizedversion of the same antibody.

The terms “polypeptide” and “protein”, used interchangeably herein,refer to a polymeric form of amino acids of any length, which caninclude coded and non-coded amino acids, chemically or biochemicallymodified or derivatized amino acids, and polypeptides having modifiedpeptide backbones.

The term “natural” antibody refers to an antibody in which the heavy andlight chains of the antibody have been made and paired by the immunesystem of a multi-cellular organism. Spleen, lymph nodes, bone marrowand serum are examples of tissues that produce natural antibodies. Forexample, the antibodies produced by the antibody producing cellsisolated from a first animal immunized with an antigen are naturalantibodies.

The term “non-naturally paired”, with respect to VH and VL chains of anengineered antibody, refers to a VH and VL pair that is not found in anatural antibody. Thus, a non-naturally paired antibody is a combinationof VH and VL chain of two different natural antibodies. The VH and VLchains of a non-naturally paired antibody are not mutated relative tothe VH and VL chains of the two different antibodies which provided theVH and VL chains. For example, the “non-naturally paired” IgH and IgLchains of the engineered antibody may contain the IgH variable chainfrom a first antibody producing cell obtained from an animal and the IgLvariable chain of second antibody producing cell obtained from the sameanimal, where the amino acid sequence of the antibody produced by thefirst cell is different from the amino acid sequence of the antibodyproduced by the second cell. In this example, the IgH and IgL chains maybe from the same lineage group. An antibody containing “non-naturallypaired” IgH and IgL chains may or not be made by phage display. As such,antibodies may or may not contain viral (e.g., bacteriophageM13)-derived sequences.

The term “lineage-related antibodies” and “antibodies that related bylineage” as well as grammatically-equivalent variants there of, areantibodies that are produced by cells that share a common B cellancestor. Related antibodies produced by related antibody producingcells bind to the same epitope of an antigen and are typically verysimilar in sequence, particularly in their L3 and H3 CDRs. Both the H3and L3 CDRs of lineage-related antibodies have an identical length and anear identical sequence (i.e., differ by up to 5, i.e., 0, 1, 2, 3, 4 or5 residues). In certain cases, the B cell ancestor contains a genomehaving a rearranged light chain VJC region and a rearranged heavy chainVDJC region, and produces an antibody that has not yet undergoneaffinity maturation. “Naïve” or “virgin” B cells present in spleentissue, are exemplary B cell common ancestors. Related antibodies arerelated via a common antibody ancestor, e.g., the antibody produced inthe naïve B cell ancestor. The term “related antibodies” is not intendedto describe a group of antibodies that are not produced by cells thatarise from the same ancestor B-cell. A “lineage group” contains a groupof antibodies that are related to one another by lineage.

The terms “treating” or “treatment” of a condition or disease refer toproviding a clinical benefit to a subject, and include: (1) preventingat least one symptom of the conditions, i.e., causing a clinical symptomto not significantly develop in a mammal that may be exposed to orpredisposed to the disease but does not yet experience or displaysymptoms of the disease, (2) inhibiting the disease, i.e., arresting orreducing the development of the disease or its symptoms, or (3)relieving the disease, i.e., causing regression of the disease or itsclinical symptoms.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

One embodiment of the subject method is illustrated in FIG. 1A. Withreference to FIG. 1A, this embodiment of the method may involveimmunizing an antibody-producing animal with a selected antigen, andenriching from a larger population of antibody-producing cells that bindto the antigen. In FIG. 1, six different antibody producing cells A-Fthat produce antibodies that bind to a target antigen are enriched froma larger population of antibody producing cells. However, in manyembodiments, there may be several hundred or several thousand enrichedcells. Each of these cells produces a natural antibody that contains anaturally paired IgH and IgL chain. The amino acid sequences of theheavy and light chains of the antibodies produced by the enriched cellsare obtained by sequencing the nucleic acids encoding the IgH and IgLchains of the antibodies, and the sequences are analyzed and put intolineage groups which, as discussed above, are groups of antibodies thatare produced by cells that share a common B cell ancestor. Suchantibodies generally have very similar sequences, and have H3 CDRs ofidentical length and near identical sequence as well as L3 CDRs ofidentical length and a near identical sequence. In the embodiment shownin FIG. 1A, the six antibody producing cells produce antibodies (AbA toAbF) that are in two lineage groups (i.e., lineage groups 1 and 2, whereAbA and AbB are in lineage group 1 and AbC, AbD, AbE and AbF are inlineage group 2). After the antibodies have been placed into lineagegroups, a single antibody (or, in certain cases, multiple antibodies(for example 2-10) from each lineage group) from at least one of thelineage group, e.g., AbA from lineage group 1 and AbC from lineage group2, is selected for testing in a bioassay, where a bioassay identifies anantibody with a biological activity (e.g., a blocking or neutralizingactivity). Once an antibody having a biological activity has beenidentified, e.g., AbC, other antibodies from the same lineage group asthe identified antibody are tested to identify a second antibody thathas the same biological activity as the first antibody. In the exampleshown in FIG. 1, antibodies D, E and F, which belong to the same lineagegroup as antibody C, were tested.

Many warm-blooded animals, in particular mammals such as humans,rabbits, mice, rats, sheep, cows, pigs and ayes such as chickens andturkeys, may be used as a source of antibody-produced cells. However, incertain embodiments a rabbit or mice is used because of their ease inhandling, well-defined genetic traits, and the fact that they may bereadily sacrificed. Procedures for immunizing animals are well known inthe art, and are described in Harlow et al., (Antibodies: A LaboratoryManual, First Edition (1988) Cold Spring Harbor, N.Y.).

Suitable antigens include extracellularly-exposed fragments of Her2,GD2, EGF-R, CEA, CD52, CD20, Lym-1, CD6, complement activating receptor(CAR), EGP40, VEGF, tumor-associated glycoprotein TAG-72 AFP(alpha-fetoprotein), BLyS (TNF and APOL—related ligand), CA125(carcinoma antigen 125), CEA (carcinoembrionic antigen), CD2 (T-cellsurface antigen), CD3 (heteromultimer associated with the TCR), CD4,CD11a (integrin alpha-L), CD14 (monocyte differentiation antigen), CD20,CD22 (B-cell receptor), CD23 (low affinity IgE receptor), CD25 (IL-2receptor alpha chain), CD30 (cytokine receptor), CD33 (myeloid cellsurface antigen), CD40 (tumor necrosis factor receptor), CD44v6(mediates adhesion of leukocytes), CD52 (CAMPATH-1), CD80 (costimulatorfor CD28 and CTLA-4), complement component C5, CTLA, EGFR, eotaxin(cytokine A11), HER2/neu, HLA-DR, HLA-DR10, HLA ClassII, IgE, GPiib/iiia(integrin), Integrin aVβ3, Integrins a4β1 and a4β7, Integrin β2,IFN-gamma, IL-1β, IL-4, IL-5, IL-6R (IL6 receptor), IL-12, IL-15, KDR(VEGFR-2), lewisy, mesothelin, MUC1, MUC18, NCAM (neural cell adhesionmolecule), oncofetal fibronectin, PDGFβR (Beta platelet-derived growthfactor receptor), PMSA, renal carcinoma antigen G250, RSV, E-Selectin,TGFbeta1, TGFbeta2, TNFalpha, TRAIL-R1, VAP-1 (vascular adhesionprotein 1) or TNFα, or the like. In many embodiments, a peptide havingthe amino acid sequence corresponding to a portion of an extracellulardomain of one of the above-listed proteins is employed as an antigen.

Antibody-producing cells may also be obtained from a subject which hasgenerated the cells during the course of a selected disease orcondition. For instance, antibody-producing cells from a human with adisease of unknown cause, such as rheumatoid arthritis, may be obtainedand used in an effort to identify antibodies which have an effect on thedisease process or which may lead to identification of an etiologicalagent or body component that is involved in the cause of the disease.Similarly, antibody-producing cells may be obtained from subjects withdisease due to known etiological agents such as malaria or AIDS. Theseantibody-producing cells may be derived from the blood, lymph nodes orbone marrow, as well as from other diseased or normal tissues.Antibody-producing cells may also be prepared from blood collected withan anticoagulant such as heparin or EDTA. The antibody-producing cellsmay be further separated from erythrocytes and polymorphs using standardprocedures such as centrifugation with Ficoll-Hypaque (Pharmacia,Uppsula, Sweden). Antibody-producing cells may also be prepared fromsolid tissues such as lymph nodes or tumors by dissociation with enzymessuch as collagenase and trypsin in the presence of EDTA.

In exemplary embodiments, an affinity purification method is utilized toisolate antibody producing cells that produce antibodies that bind to anantigen. The antigen with which the animal was immunized may beimmobilized on a solid phase and used to selectively retain antibodyproducing cells that express an antibody on their surface that binds tothe antigen, while other cells are washed away. The retained cells maythen be eluted by a variety of methods, such as by using an excess ofthe antigen, chaotropic agents, changing the pH, salt concentration,etc. Any of the well known methods for immobilizing or coupling antigento a solid phase may be used. For example, when the antigen is a cancercell, appropriately treated microtiter plate that will bind to cells maybe used, such as microtiter plates for cell culture. In the instanceswhere the antigen is a protein, the protein may be covalently attachedto a solid phase, for example, sepharose beads, by well knowntechniques, etc. Alternatively, a labeled antigen may be used tospecifically label cells that express an antibody that binds to theantigen and the labeled cells may then be isolated by cell sorting(e.g., by FACS). In certain cases, methods for antibody purification maybe adapted to isolate antibody producing cells. Such methods are wellknown and are described in, for example, J Immunol Methods. 2003November; 282(1-2):45-52; J Chromatogr A. 2007 Aug. 10; 1160(1-2):44-55;J Biochem Biophys Methods. 2002 May 31; 51 (3):217-31. Cells may also beisolated using magnetic beads or by any other affinity solid phasecapture method, protocols for which are known. In some embodiments,antigen-specific antibody producing cells may be obtained from blood byflow cytometry using the methods described in Wrammert (Nature 2008 453:667-672), Scheid (Nature 2009 458: 636-640), Tiller (J. Immunol. Methods2008 329 112-124) or Scheid (Proc. Natl. Acad. Sci. 2008 105:9727-9732), for example, which are incorporated by reference fordisclosure of those methods. Exemplary antibody-producing cellenrichment methods include performing flow cytometry (FACS) of cellpopulations obtained from a spleen, bone marrow, lymph node or otherlymph organs, e.g., through incubating the cells with labeledanti-rabbit IgG and sorting the labeled cells using a FACSVantage SEcell sorter (Becton-Dickinson, San Jose, Calif.). In some embodiments,single or nearly single antibody-producing cells are deposited inmicrotiter plates. If the FACS system is employed, sorted cells may bedeposited after enrichment directly into a microtiter plate.

Enrichment may decrease the size of the cell population by at least 50%,e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least95% or at least 99% and in certain cases, the plurality of enrichedantibody producing cells may be substantially pure, i.e., substantiallyfree of other cells that do not produce an antibody that binds to theantigen, where the term “substantially pure” refers to an isolatedpopulation of antibody producing cells, in which cells that expressantibodies that specifically bind to the antigen make up at least 5%,10%, 20%, 30%, at least 40%, at least 50%, at least 60%, at least 70% ormore of the total population of cells. The enriched population ofantibody producing cells may be employed as a mixture of cells, oralternatively, they may be used as single cells, e.g., by dilution anddeposition into individual wells of a microtiter plate.

The enriched population of antibody producing cells may comprise atleast 5, at least 10, at least 30, at least 60, at least 100, at least300, at least 500, at least 1000, at least 5,000, at least 10,000 or atleast 100,000, or more antibody producing cells.

The isolated antibody-producing cells may be optionally cultured (i.e.grown in media that supports at least one, at least 5 or at least 10 ormore cell divisions of the cell) by methods known to one of skill in theart after they have been deposited (see e.g. WO 01/55216).

In certain embodiments, the antibodies produced by the enriched cellsare not well characterized. As such, although the antibody-producingcells are isolated based on the production of antibodies thatspecifically bind to the antigen, the epitope(s) to which theseantibodies bind is unknown, and it is not known if the antibodies haveany biological activity (e.g., a neutralizing or blocking activity).Additionally, the nucleic acid sequence or the amino acid sequence ofthe variable regions of IgH and IgL chains of these antibodies are notknown.

Sequences encoding heavy and light chains may be amplified from the cDNAusing techniques well known in the art, such as Polymerase ChainReaction (PCR). See Mullis, U.S. Pat. No. 4,683,195; Mullis et al., U.S.Pat. No. 4,683,195; Polymerase Chain Reaction: Current Communication inMolecular Biology, Cold Springs Harbor Press, Cold Spring Harbor, N.Y.,1989. Briefly, cDNA segments encoding the variable domain of theantibody are exponentially amplified by performing sequential reactionswith a DNA polymerase. The reaction is primed by a 5′ primer and a 3′DNA primer. In some embodiments, the 3′ antisense primer correspondingto a DNA sequence in the constant (or joining) region of theimmunoglobulin chain and the 5′ primer (or panel of related primers)corresponding to a DNA sequence in the variable region of theimmunoglobulin chain. This combination of oligonucleotide primers hasbeen used in the PCR amplification of murine immunoglobulin cDNAs ofunknown sequence (see Sastry et at., Proc Natl. Acad. Sci. 86:5728-5732,1989 and Orlandi et al., Proc. Natl. Acad. Sci. 86:3833-3837, 1989).Alternatively, an “anchored polymerase chain reaction” may be performed(see Loh et al., Science 243:217-220, 1989). In this procedure, thefirst strand cDNA is primed with a 3′ DNA primer as above, and a poly(dGtail) is then added to the 3′ end of the strand with terminaldeoxynucleotidyl transferase. The product is then amplified by PCR usingthe specific 3′ DNA primer and another oligonucleotide consisting of apoly(dC) tail attached to a sequence with convenient restriction sites.In many embodiments, however, the entire polynucleotide encoding a heavyor light chain is amplified using primers spanning the start codons andstop codons of both of the immunoglobulin cDNAs, however, depending onthe amplification products desired, suitable primers may be used.Exemplary primers for use with rabbit antibody-producing cells are asfollows: heavy chain, 5′ end(CACCATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTG; SEQ ID NO:177); heavychain, 3′ end (CTCCCGCTCTCCGGGTAAATGAGCGCTGTGCCGGCGA; SEQ ID NO:178);light chain kappa, 5′end (CAGGCAGGACCCAGCATGGACACGAGGGCCCCCACT; SEQ IDNO:179); and L kappa, 3′end (TCAATAGGGGTGACTGTTAGAGCGAGACGCCTGC; SEQ IDNO:180). Suitable restriction sites and other tails may be engineeredinto the amplification oligonucleotides to facilitate cloning andfurther processing of the amplification products. Amplificationprocedures using nested primers may also be used, where such nestedprimers are well known to one of skill in the art. Exemplary methods foramplifying antibody-encoding nucleic acid is also described in Wrammert(Nature 2008 453: 667-672) and Scheid (Nature 2009 458: 636-640), forexample. In this embodiment, the enriched cells may be combined beforesequencing (in which case the initial amplification product will containa mixture of a plurality of different products that can be discriminatedby cloning the products or using single molecule sequencingtechnologies), or the cells may be kept separate from one another (inwhich case the initial amplification product amplified from a singlecell may contain a single species that can be sequenced).

In certain embodiments, at least 1,000 heavy chain sequences and atleast 1,000 light chain sequences are obtained.

Once the amino acid sequence of heavy and light chains of the antibodieshas been obtained, antibodies can be grouped on the basis of sequencesimilarity to provide a plurality of groups of antibodies that arerelated by lineage. Methods for performing clonal analysis of antibodysequences are well known and are described in a number of publicationsincluding Magori-Cohen (Bioinformatics 2006 22: e332-40), Manske (Clin.Immunol. 2006 120:106-20), Kleinstein (J. Immunol. 2003 171: 4639-49),Clement (Mol. Ecol. 2000 9: 1657-1659), Mehr (J. Immunol. 2004 1724790-6), Wrammert (Nature 2008 453: 667-672), Scheid (Nature 2009 458:636-640), which are incorporated by reference herein for disclosure ofthose methods. The antibodies placed into lineage groups should all befrom a single animal, i.e., an individual mouse or rabbit.

In some embodiments, the amino acid positions of an antibody arenumbered using a suitable numbering system, such as that provided byChothia (J Mol Biol 1998; 278: 457-79) or Kabat (1991, Sequences ofProteins of Immunological Interest, DHHS, Washington, D.C.). CDR and/orframework residues may be identified using these methods. The numberedsequences may be aligned by eye, or by employing an alignment programsuch as one of the CLUSTAL suite of programs (Thompson et al NucleicAcids Research, 22:4673-4680). The variable regions of antibodies withina related group of antibodies have amino acid sequences that are verysimilar. For example, the VH or VL domains of antibodies within arelated group of antibodies may have amino acid sequences that are atleast about 80% identical (e.g., at least 85% identical, at least 90%identical, at least 95% or at least 98% or at least 99% identical),ignoring any gaps or insertions made to facilitate alignment of thesequences. Antibodies within a related group of antibodies have a VLdomains that are similar to each other, as well as VH domains that aresimilar to each other. In other words, in certain embodiments the VH orVL domains of two different related antibodies usually contain up toabout ten (i.e., one, two, three, four or five or more) amino aciddifferences. An amino acid difference may be present at any position ofthe variable domain, including in any CDR or in any framework region.Certain related antibodies have H3 CDRs that are almost identical, aswell as L3 CDRs that are almost identical. In these embodiments, any twoantibodies that are related will have L3 and H3 CDRs that are eachidentical in length and have near identical sequences (i.e., thatcontain 0, 1, 2, 3, 4 or 5 amino acid changes). In other words the L3CDRs of the two antibodies are identical in length and near identical insequence and the H3 CDRs of the two antibodies are identical in lengthand near identical in sequence. Two exemplary sets of related antibodiesare shown in FIG. 2, and the sequences of 20 exemplary VH3 regions ofunrelated rabbit antibodies are shown for comparison in FIG. 3.

In certain embodiments, the heavy chain sequences may or may not begrouped independently of the light chain sequences. If the heavy andlight chain sequences are grouped independently of one another, theheavy and light chain groups may be matched up by analysis of lineagetrees.

Depending how many sequences are obtained, in certain embodiments theenriched antibodies may be grouped into at least 5 groups, at least 10groups, at least 20 groups, at least 50 groups, or at least 100 groupsor more, e.g., up to 200 or 500 groups or more. Depending how manysequences are obtained, each group may contain from 2 to several hundredor more antibodies.

Once the antibodies have been grouped, a single antibody from each of atleast some of the groups (e.g., at least 20%, at least 50 or at least80% of the groups) is tested in a first bioassay to identify a firstantibody that has a biological activity. The bioassay may determinewhether the antibody has a biological effect, e.g., an ability toinhibit an interaction between a receptor and an a ligand by eitherbinding to the receptor and blocking binding of the ligand, or bybinding to the ligand and neutralizing it, or by promoting or inhibitinga cellular phenotype, e.g., cell growth, cell proliferation, cellmigration, cell viability (e.g., apoptosis), cell differentiation, celladherence, cell shape changes (e.g., tubular cell formation), complementdependant cytotoxicity CDC, antibody-dependent cell-mediatedcytotoxicity ADCC, receptor activation, gene expression changes, changesin post-translational modification (e.g., phosphorylatoin), changes inprotein targeting (e.g., NFκB localization etc.), etc., or inhibition ofreceptor multimerization (e.g., dimer or trimerization) orreceptor-ligand interactions, etc. Such bioassays are well known in theart. The term “bioassay” is intended to exclude assays in which only theability of an antibody to bind to a target is read. Bioassays useful inthis method are numerous, and include but are not limited to cellularassays in which a cellular phenotype is measured, e.g., gene expressionassays; and in vivo assays that involve a particular animal (which, incertain embodiments may be an animal model for a condition related tothe target). In certain cases, the assay may be a vascularization assay.

In this embodiment, the antibodies tested in the bioassay may containnaturally paired heavy and light chain variable domains, ornon-naturally paired heavy and light chains (i.e., heavy and light chainvariable domains from different antibodies of the same lineage group).Since the antibodies are from the same lineage group, it is expectedthat such antibodies will be functional.

After a first antibody that has a biological activity has beenidentified, further antibodies that are in the same lineage group as thefirst antibody are tested in a second bioassay, thereby identifying asecond antibody that has the same biological activity as the firstantibody. In certain cases at least 10%, at least 20%, at least 50%, orat least 80% of the antibodies in the same lineage group are tested. Thefirst bioassay may be the same as or different to the second bioassay.In certain embodiments, a plurality of antibodies is tested, and theantibody with the best properties is chosen for future use.

In particular embodiments, the further antibodies may contain naturallypaired heavy and light chain variable domains, or non-naturally pairedheavy and light chain variable domains (i.e., heavy and light chainvariable domains from different antibodies of the same lineage group).Since the antibodies are from the same lineage group, it is expectedthat such antibodies will be functional. In particular embodiments, thepairing of the heavy and light chains may be systematic (e.g., everyheavy chain is tested in combination with every light chain) or random(e.g., every heavy chain is tested with randomly selected light chains),for example.

Exemplary VEGF bioassays include assays using isolated protein in a cellfree systems, in vitro using cultured cells or in vivo assays. ExemplaryVEGF assays include, but are not limited to a receptor tyrosine kinaseinhibition assay (see, e.g., Cancer Research Jun. 15, 2006;66:6025-6032), an in vitro HUVEC proliferation assay (FASEB Journal2006; 20: 2027-2035), an in vivo solid tumor disease assay (U.S. Pat.No. 6,811,779) and an in vivo angiogenesis assay (FASEB Journal 2006;20: 2027-2035). These assays are well known in the art. The descriptionsof these assays are hereby incorporated by reference.

Exemplary TNF-α bioassays include in vitro assays using cell freesystems or using cultured cells or in vivo assays. As such, TNF-α assaysinclude in vitro human whole blood assay and cell mediated cytotoxicityassay (U.S. Pat. No. 6,090,382), in vitro tumor human killing assay(see, e.g., published U.S. patent application 20040185047), in vivotumor regression assay (USP Application 20040002589). Additional TNF-αassays are described in a variety of publications, including20040151722, 20050037008, 20040185047, 20040138427, 20030187231,20030199679, and Balazovich (Blood 1996 88: 690-696).

A subject antibody inhibits at least one activity of its target in therange of about 20% to 100%, e.g., by at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, usuallyup to about 70%, up to about 80%, up to about 90% or more. In certainassays, a subject antibody may inhibits its target with an IC₅₀ of1×10⁻⁷ M or less (e.g., 1×10⁻⁷ M or less, 1×10⁻⁸ M or less, 1×10⁻⁹ M orless, usually to 1×10⁻¹² M or 1×10⁻¹³ M). In assays in which a mouse isemployed, a subject antibody may have an ED₅₀ of less then 1 μg/mouse(e.g., 10 ng/mouse to about 1 μg/mouse). In certain embodiments, asubject antibody may be contacted with a cell in the presence of aligand, and a ligand response phenotype of the cell is monitored.

In certain embodiments, particularly if the antigen elicits a strongresponse in the animal, the method may be practiced in the absence ofany antigen-based enrichment of antibody producing cells prior to thefirst bioassay. In these embodiments, the method may involve: a)obtaining the antibody heavy chain sequences and the antibody lightchain sequences from a population of B cells of an animal, wherein saidpopulation of B cells is not enriched for B cells that produceantibodies that specifically bind to a target antigen, b) grouping theheavy and light chain sequences on the basis of sequence similarity toprovide a plurality of groups of antibodies that are related by lineage;c) testing a single antibody from each of the groups in a first bioassayto identify a first antibody that has a biological activity; and, afterthe first antibody has been identified; and d) testing furtherantibodies that are in the same group as the first antibody in a secondbioassay, thereby identifying a second antibody that has the biologicalactivity.

Another embodiment of is illustrated in FIG. 1B. With reference to FIG.1B, this embodiment of the subject method may involve immunizing anantibody-producing animal with a selected antigen, and testing aplurality of antibodies produced by a first portion of an antibodyproducing organ of the animal (e.g., a first portion of the spleen, afirst portion of the lymph nodes, a first portion of bone marrow, or afirst portion of the peripheral blood mononuclear cell (PBMC) populationin the bloodstream of the animal, etc.) in a bioassay to identify afirst antibody that has a biological activity. In this embodiment, thefirst and second portions of an antibody-producing organ need not bespatially separated in the organ. Rather, since a first portion or anorgan can be made by, for example, making a single cell suspension ofthe organ and then removing part of the suspension, the first and secondportions of an organ may be interspersed with one another in the organ.In the example shown in FIG. 1B, antibody A is identified as having abiological activity. The nucleotide sequence encoding the IgH and IgLchain of the antibody is obtained. Based on these sequences, PCR primersthat are specific for the heavy and light chains of antibodies that arein the same lineage group as the identified antibody are designed, andused to obtain from the second portion of the antibody producing organthe sequences of further antibodies that are in the same lineage groupas the identified antibody. The further antibodies are tested, and asecond antibody from the same lineage group and also having the samebiological activity as the first antibody is identified.

Many exemplary aspects of this alternative method, e.g., which antigensand bioassays can be employed in the method, etc., are discussed above.In certain embodiments, a lead antibody obtained from a first portion ofan antibody-producing organ is identified using a bioassay. In thisembodiment, the antibodies obtained from the first portion of the organare screened using a hybridoma-based method or by a method that does notrequire production of hybridomas, e.g., by phage display or by themethod described in US20040067496 and other references, for example, toidentify a biologically active antibody. In one embodiment, a portion ofthe splenocytes of a spleen of a single animal is fused with a fusionpartner to produce hybridomas that are then screened to identify abiologically active antibody. In another embodiment, heavy and lightchain sequences are directly amplified from PBMCs, and recombinantantibodies are expressed in a different cell (e.g., as described inUS20040067496) prior to screening. In another embodiment, a phagedisplay library is constructed from the RNA made from a portion of thespleen of an animal, and the phage display library is screened. Thefirst, biologically active antibody is identified, and the nucleic acidencoding that antibody is sequenced.

In certain embodiments, polynucleotides encoding the variable heavy andvariable light domains of lineage-related antibodies may be amplifiedfrom the same animal as the first antibody by “CDR-anchored PCR”, i.e.,using pairs of primers that each contains a primer that is complementaryto a CDR-encoding region of the parent antibody cDNA. In theseembodiments, the method may include: a) obtaining the nucleotidesequences of: i. a heavy chain-encoding nucleic acid that encodes thevariable heavy chain of a first antibody of an immunized animal; and ii.a variable light chain-encoding nucleic acid that encodes the lightchain of the first antibody; b) obtaining the amino acid sequence of thevariable domains of the heavy and light chains of further antibodiesfrom the immunized animal, using: i. a first primer pair that includes afirst primer that is complementary to a CDR-encoding region of the heavychain-encoding nucleic acid; and ii. a second primer pair that includesa second primer that is complementary to a CDR-encoding region of thelight chain-encoding nucleic acid. After the amino acid sequences of thevariable domains of the further antibodies have been determined bytranslation of the obtained nucleotide sequences, the amino acid may beanalyzed using the above methods to confirm that they are related bylineage to the first antibody (e.g., analyzed to determine whether theamino acid sequences of the heavy and light chains are at least 80%identical to those of the parent antibody and whether the heavy andlight chain CDR3 regions are of identical length of near identicalsequence etc. as discussed above).

As would be readily apparent, a variety of techniques are available foramplifying sequences that encode further antibodies from an animal afterthe nucleotide sequence encoding a first antibody has been obtained fromthat animal. For example, sequences encoding the heavy and light chainsof the second antibody may be amplified using inverse PCR (e.g., usingtwo primers that face away from each other) or by anchored PCR using aspecific (where a specific primer may be complementary to a differentsequence of the first antibody, e.g., a different CDR sequence) or“universal” primer (where a universal primer is complementary to asequence that is present in a plurality of different antibody-encodingpolynucleotides), where one of the primers is complementary to firstCDR-encoding region using cDNA as a template. In certain cases, auniversal primer may be complementary to a sequence that is in at least10% (e.g., at least 20% at least 40% at least 50% or at least 80%) ofall heavy or light chain encoding cDNAs obtainable from the animal(e.g., complementary to nucleic acid encoding a conserved sequence thatis present in the constant region or secretion signal of theantibodies). In other embodiments, the universal primer may becomplementary to flanking sequences in the vector into which cDNA fromthe animal is cloned or to linkers ligated onto the cDNA, for example.

In one embodiment, two amplification reactions are performed using cDNAas a template, where the first reaction amplifies the heavy chainvariable domain-encoding nucleic acid for the second antibody and thesecond reaction amplifies the light chain variable domain-encodingnucleic acid for the second antibody. In this embodiment: a) the firstreaction uses: i. a CDR-specific primer that is complementary to aCDR-encoding region (i.e., the CDR1, CDR2 or CDR3 region) of the heavychain-encoding nucleic acid of the first antibody and ii. a universalsecond primer that is complementary to a non-variable domain-encodingregion of the antibody heavy chain cDNA, e.g., to a sequence thatencodes the constant domain or secretion signal of the heavy chain ofthe first antibody, as illustrated in the examples section of thisdisclosure; and b) the second reaction uses i. a CDR-specific primerthat is complementary to a CDR-encoding region (i.e., the CDR1, CDR2 orCDR3 region) of the light chain-encoding nucleic acid of the firstantibody and ii. a universal second primer that is complementary to anon-variable domain-encoding region of the antibody light chain cDNA,e.g., to a sequence that encodes the constant domain or secretion signalof the light chain of the first antibody, as illustrated in the examplessection of this disclosure.

Several strategies for cloning antibody sequences by PCR are known andmay be readily adapted for use in the instant method (e.g., by using aCDR-specific primer in addition to a disclosed primer). Such strategiesinclude those described by: LeBoeuf (Cloning and sequencing ofimmunoglobulin variable-region genes using degenerateoligodeoxyribonucleotides and polymerase chain reaction. Gene. 198982:371-7), Dattamajumdar (Rapid cloning of any rearranged mouseimmunoglobulin variable genes Immunogenetics. 1996 43:141-51),Kettleborough (Optimization of primers for cloning libraries of mouseimmunoglobulin genes using the polymerase chain reaction Eur. J.Immunol. 1993 23:206-11), Babcook (A novel strategy for generatingmonoclonal antibodies from single, isolated lymphocytes producingantibodies of defined specificities Proc. Natl. Acad. Sci. 1996 93:7843-7848) and Williams (Structural diversity in domains of theimmunoglobulin superfamily. Cold Spring Harb. Symp. Quant. Biol. 198954:637-47) as well as many others. In certain cases, the second primermay be a mixture of different primers or degenerate primers, forexample.

The heavy chain CDR-specific primer may be complementary to the sequencethat encodes the CDR1, CDR2 or CDR3 region of the heavy chain of thefirst antibody and, likewise, the light chain CDR-specific primer may becomplementary to the sequence that encodes the CDR1, CDR2 or CDR3 regionof the light chain of the first antibody. In certain embodiments, aparticular CDR-specific primer may be chosen because the CDR sequence towhich it binds may be known to be less variable than other CDRsequences.

Such CDR-anchored amplification method described in U.S. patentapplication Ser. No. 61/151,052, filed Feb. 9, 2009, which isincorporated by reference in its entirety for disclosure of thosemethods.

The above-described CDR-anchored method is effective because mostsequence diversity between the variable domains in different families ofantibodies that are related by lineage is in the CDR regions (i.e., theCDRs are quite variable between different families of antibodies),whereas the sequence of the CDR regions is relatively constant withinthe antibodies of a single family of antibodies that are related bylineage. Because the method uses primers that are complementary tosequence that are highly variable between different families of relatedantibodies, only related antibodies should be successfully amplified bythe method.

In this embodiment, an amplification reaction may be performed usingcDNA made from a second portion of the antibody-producing organ. Forexample, the amplification reaction may be done using nucleic acidobtained from single cells (or cultures of the same) or nucleic acidobtained from pooled cells (e.g., pools of different antibody-producingcells that each contain cDNA). Pools may contain cDNA from at least 10,at least 100 or at least 1,000 different antibody cells, for example. Inembodiments in which hybridomas are used, the identity of the hybridomasthat contributed to each pool may be tracked in order to identify ahybridoma producing a second antibody if the sequence encoding thesecond antibody is successfully amplified. Amplification products of theexpected size may be sequenced directly or cloned and sequenced usingknown methods.

Depending on the antigen and number of antibody-producing cells in thesecond portion of the antibody-producing organ, the heavy and lightchain variable sequences for at least 5, at least 10, at least 20, atleast 50 or at least 100 or more, e.g., up to 200, up to 500, 1,000,5,000 or 10,000 or more sequences may be obtained.

The further antibodies are tested in a second bioassay to identify asecond antibody that has the same biological activity as the firstantibody. As noted above, the first and second bioassays may be the sameor different. In certain cases at least 30% (e.g., at least 70%, atleast 80%, or at least 90%) of the lineage-related antibodies are testedin the bioassay. In this embodiment, the further antibodies may containnaturally paired heavy and light chain variable domains, ornon-naturally paired heavy and light chains (i.e., heavy and light chainvariable domains from different antibodies of the same lineage group).Since the antibodies are from the same lineage group, it is expectedthat such antibodies will be functional. In particular embodiments, thepairing of the heavy and light chains may be systematic (e.g., everyheavy chain is tested in combination with every light chain) or random(e.g., every heavy chain is tested with randomly selected light chains),for example.

An antibody produced by the instant methods finds use in diagnostics, inantibody imaging, and in treating diseases treatable by monoclonalantibody-based therapy. In particular, an antibody humanized by theinstant methods may be used for passive immunization or the removal ofunwanted cells or antigens, such as by complement mediated lysis orantibody mediated cytotoxicity (ADCC), all without substantial immunereactions (e.g., anaphylactic shock) associated with many priorantibodies. For example, the antibodies of the present invention may beused as a treatment for a disease where the surface of an unwanted cellspecifically expresses a protein recognized the antibody (e.g. HER2, orany other cancer-specific marker) or the antibodies may be used toneutralize an undesirable toxin, irritant or pathogen. Humanizedantibodies are particularly useful for the treatment of many types ofcancer, for example colon cancer, lung cancer, breast cancer prostatecancer, etc., where the cancers are associated with expression of aparticular cellular marker. Since most, if not all, disease-relatedcells and pathogens have molecular markers that are potential targetsfor antibodies, many diseases are potential indications for humanizedantibodies. These include autoimmune diseases where a particular type ofimmune cells attack self-antigens, such as insulin-dependent diabetesmellitus, systemic lupus erythematosus, pernicious anemia, allergy andrheumatoid arthritis; transplantation related immune activation, such asgraft rejection and graft-vs-host disease; other immune system diseasessuch as septic shock; infectious diseases, such as viral infection orbacteria infection; cardiovascular diseases such as thrombosis andneurological diseases such as Alzheimer's disease.

An antibody of particular interest is one that modulates, i.e., reducesor increases a symptom of the animal model disease or condition by atleast about 10%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 80%, at least about 90%, ormore, when compared to a control in the absence of the antibody. Ingeneral, a monoclonal antibody of interest will cause a subject animalto be more similar to an equivalent animal that is not suffering fromthe disease or condition. Monoclonal antibodies that have therapeuticvalue that have been identified using the methods and compositions ofthe invention are termed “therapeutic” antibodies.

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain embodiments and aspects of the present invention andare not to be construed as limiting the scope thereof.

Example 1 Method of Producing a Library of Engineered-Antibody ProducingCells

Isolation of Antibody Producing Cells

Rabbits are immunized with an antigen using a standard immunizationprotocol. At about 10 days after the second booster immunization,antibody titers are determined using ELISA. Two booster immunizationsare usually sufficient for obtaining high antibody titers. As soon as ahigh titer (detectable signal at 1:100000 dilution) is observed, therabbit is sacrificed and bone marrow cells are collected from the femurand/or other large bones. Spleen cells and peripheral blood mononuclearcells (PBMCs) are also collected and frozen in 10% DMSO/90% FBS foranalysis at a later time. Very large numbers of bone marrow cells (>2billion) are obtained from a single rabbit. After washing, clearing ofdebris, and red-cell lysis, the antibody producing cells, which bind tothe antigen with which the rabbit was immunized, are purified usingFACS. Briefly, the antigen is conjugated to a fluorescent dye and thelabeled antigen is incubated with the cells obtained above. The cellsare briefly rinsed to wash off any antigen non-specifically attached tothe cell. After rinsing, fluorescent cells are separated from unlabeledcells using FACS. These fluorescent cells express antibodies on theirsurface that specifically binds to the antigen with which the animal wasimmunized.

RT-PCR to Obtain IgH and IgL Chain cDNA

Primer Design:

In rabbit, the 5′ coding sequences of rabbit immunoglobulin heavy chainare primarily derived from only one gene. Antibody diversity is createdby gene conversion and somatic mutation, but this does not affect the 5′end of the antibody cDNA. Thus, most rabbit IgG H chains have verysimilar or identical signal peptide sequences, and the same is true forL chains. On the 3′ side, primers hybridizing to the constant domains,which also have identical sequences in most rabbit antibodies (rabbitconstant domains are not divided into subclasses). As a result, only onepair of primers each is required for amplifying the vast majority ofrabbit IgG H and L sequences. Typical priming sites are shown below,although any primer sites are used so long as the a variabledomain-encoding polynucleotide is amplified. Typical primers for usewith rabbit antibody-producing cells are as follows: heavy chain, 5′ end(CACCATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTG (SEQ ID NO: 181)); heavychain, 3′ end (CTCCCGCTCTCCGGGTAAATGAGCGCTGTGCCGGCGA (SEQ ID NO: 182));light chain kappa, 5′end (CAGGCAGGACCCAGCATGGACACGAGGGCCCCCACT (SEQ IDNO: 183)); and L kappa, 3′end (TCAATAGGGGTGACTGTTAGAGCGAGACGCCTGC(SEQ IDNO: 184)).

Note that the 3′ H chain primer spans the 3′ end of the coding region,the stop codon, and the beginning of the 3′ UTR. Thus, this primer isspecific for the secreted form of IgG, and does not recognize thetransmembrane form, which does not contain this sequence due toalternative splicing. Therefore, the method is unlikely to recover IgGfrom memory B cells, which express predominantly the transmembrane form.

RT-PCR Conditions:

Cell lysis is done heating in a buffer containing RNAse inhibitors,followed by DNA degradation and reverse transcription performed at hightemperature (60° C.) using a thermostable reverse transcriptase. Reversetranscription is primed by primers specific for the 3′ region of the IgGmRNAs. A single-step RT-PCR protocol is used, utilizing a thermostableenzyme that has both reverse transcriptase and DNA polymerase activities(MasterAmp™ RT-PCR Kit for High Sensitivity, Epicentre Technologies,Madison, Wis.). PCR products are analyzed by agarose gelelectrophoresis. If required, a second round of PCR is performed withnested primers. In some PCR applications, this step is required toproduce sufficient amounts of specific product.

Co-Amplification of H and L Chain cDNAs:

Different combinations of primers are tried, to accomplish efficient PCRamplification of H and L chain cDNAs in the same reaction. A ‘headstart’ approach is often used, where PCR cycling is started with H chainprimers alone; after a number of cycles (5 to 10) the L chain primersare added to the mix. Using these methods, similar yields of H and Lchain are produced. Alternatively, a nested PCR approach is used for theH chain, by performing an initial round of PCR with primers amplifyingthe full-length cDNA, and a second round with primers amplifying onlythe vH-cH1-hinge portion of the H chain. This method should yield aproduct similar in size to the L chain cDNA. Expression of this productyields the F(ab′)2 fragment of IgG, which is divalent and fully activefor antigen-binding.

IgG heavy and light chain PCR products are joined with CMV promoter andBGH3′pA (bovine growth hormone polyadenylation/transcriptiontermination) sequences.

Method a) Overlap Extension PCR.

CMV Promoter Segment:

To prepare the CMV promoter fragment, the expression vector pcDNA-3(which contains the CMV promoter and BGH3′pA segments) is used as atemplate, and the following PCR setup:

Primer 1 (5′ AATTCACATTGATTATTGAG 3′; SEQ ID NO: 185) corresponding tothe 5′ end of the CMV promoter;

Primer 2 (5′ CAGCGCAGCCCAGTCTCCATCCCGTAAGCAGTGGGTTCTC 3′; SEQ ID NO:186) corresponding to the 3′ end of the CMV promoter, and containing a5′ extension (underlined) complementary to the 5′ end of the rabbit Ig Hsignal peptide sequence is performed.

PCR amplification with these primers produces a linear DNA fragmentconsisting of the CMV promoter (610 nt) and a 20 nt extension on the 3′end, which is complementary to the 5′ end of the IgG vH coding region.As mentioned above, most rabbit IgGs contain 5′ vH (signal peptide)regions with nearly identical sequences. Therefore, only one primer pairis needed to amplify the majority of rabbit IgG cDNAs.

BGH3′pA Segment.

A similar approach is used to prepare the BGH3′pA segment. Again, thepcDNA3 expression vector is used as a template, and the followingprimers are used:

Primer 3 (5′ CCGGGTAAATGAGCGCTGTGGTTTAAACCCGCTGATCAGC 3′; SEQ ID NO:187), corresponding to 5′ end of the BGH3′pA domain extended by a 20 ntsequence complementary to the 3′ end of the IgG heavy chain codingregion, and including 11 nt of the 3′ untranslated domain.

Primer 4 (5′ AAGCCATAGAGCCGACCGCA 3′; SEQ ID NO: 188) corresponding tothe 3′ end of the BGH polyadenylation domain.

PCR amplification results in a 250 nt fragment containing the BGH3′pAsequence and a 20 nt extension that overlaps with the 3′ end of the IgGheavy chain sequence.

Overlay Extension PCR:

The IgG heavy chain PCR product are mixed with the CMV promoter andBGH3′pA segments. The mixture is subjected to 10 cycles of PCR.

The overlapping segments anneal, followed by extension of theoverlapping 3′ ends. At the end of the 10 cycles, the outside primers(primers 1 and 4) are added to the mixture, and another 30 cycles of PCRare performed. The product is a 2100 nt fragment consisting of the CMVpromoter, the IgG H coding sequence, and the BGH terminator.

IgG Light Chain:

The process are carried out in an analogous manner to produce 1500 ntfragments consisting of CMV promoter, kappa light chain coding sequence,and BGH terminator. A separate set of primers for lambda light chainscan also be used to amplify and clone lambda light chains.

A low concentration of primers in the initial PCR reaction may be used.In some embodiments, primers are be designed such that amplification ofthe heavy chain results in a nucleotide encoding a form of the IgG Hchain that is truncated at the 3′ end of the hinge domain. This fragmentwould be similar in size to the v kappa light chain. Co-expression ofthese fragments results in the secretion of F (ab′)₂ fragments of IgG.

Method b) Topoisomerase I Coupling.

This method is used as an alternative to overlap extension PCR. Theoverall experimental strategy is as described above. Commerciallyavailable topoisomerase-modified CMV promoter and BGH3′pA segments willbe used (Invitrogen, San Diego, Calif.). The CMV promoter element (610nt) is provided in a modified form with the topoisomerase recognitionsite (CCCTT) at its 3′ end, and a six base pair single-stranded overhangat the 3′ end (GCCTTG) which is used for directional coupling with thePCR product. The topoisomerase I enzyme is bound to the recognition siteCCCTT. In order to be joined to the Topo-modified CMV promoter, the PCRproduct needs to contain the sequence CGGAACAAGGG (SEQ ID NO: 189) atits 5′end. This sequence is cleaved by topoisomerase, resulting in a6-base single-strand overhang that is complementary to the single-strandoverhang of the CMV promoter element. These overhangs anneal and thefragments are covalently joined by the enzyme.

In order to link the IgG cDNA fragment to the CMV promoter, the 5′primer used in the last round of IgG amplification are extended at its5′ end with the sequence CGGAACAAGGG (SEQ ID NO: 190).

The linkage of the 3′ end of the IgG fragment with the BGH3′pA elementis performed in an analogous manner, except that a differentsingle-stranded overhang (GACTCA) is being used. This provides fordirectionality and selective joining of the 5′ end with the CMV promoterand the 3′ with the BGH terminator.

The joining reaction is carried out by mixing the 5′ CMV element, IgGPCR product, and 3′BGH element at a 1:2:1 ratio, and adding the 10×reaction buffer. The reaction proceeds rapidly and is usually completewithin 10 min at room temperature. Following the reaction, a secondaryPCR reaction is carried out, using primers corresponding to the 5′ endof the CMV promoter and the 3′ end of the BGH terminator (primers 1 and4, see above). This results in the formation of the 2.1 kb IgG Hexpression cassette, or the 1.5 kb IgG L expression cassette. Conditionsfor co-production of H and L IgG expression cassettes in the samereaction are also envisioned.

The IgG H expression cassettes are cloned into a vector carrying ahygromycin resistance marker to generate an IgG H expression cassettelibrary. The IgG L expression cassettes are cloned into a vectorcarrying a G418 resistance marker to generate an IgG L expressioncassette library.

Equimolar amounts of the IgG H and IgG L expression cassette librariesare mixed and transfected into CHO cells. The transfected CHO cells areplated into 96-well or 384-well microtiter plates such that each wellcontains approximately one cell. Cells are maintained in mediacontaining both hygromycin and G418. Cells that survive the doubleselection contain at least one expression cassette pair.

These cells are cultured and the antibodies produced by these cells aretested for binding to the antigen with which the rabbit was immunized.

Example 2 Related Antibodies

Antibodies were obtained from rabbit hybridoma cells producing anti-KDRantibodies that block the interaction of VEGF with its receptor (KDR).The hybridoma cells were generated by fusing immunized rabbitsplenocytes with the rabbit hybridoma fusion partner 240E-W2.

New Zealand white rabbits were immunized with a fusion proteincontaining the rabbit Fc region and the extracellular domain of KDR.Each rabbit received a primary immunization by subcutaneous injection of0.4 mg of the purified protein with complete Freund's or TiterMaxadjuvant. The animals were then boosted by subcutaneous injection of 0.2mg of the protein with incomplete Freund's or TiterMax once every threeweeks. The final boost (0.4 mg protein in saline) was givenintravenously 4 days before splenectomy.

Cell fusions were performed following the conventional protocol ofSpieker-Polet using PEG. The ratio of splenocytes to the fusion partnerwas 2:1. The fused cells were plated in 96-well plates and HAT was addedafter 48 hrs to select for hybridomas. Direct ELISA was performed toidentify antibodies that block binding of VEGF to a KDR fusion proteincoated onto a microtiter plate. In this assay, the Fc-KDR ECD fusionprotein was coated onto a 96-well ELISA plate and goat anti-rabbit IgGFEB conjugated to alkaline phosphatase was used to detect antibodybinding to KDR. Antibodies identified in this assay were then werescreened for blocking VEGF interaction with KDR in a ligand-receptorassay. The blocking antibodies were identified by their inhibition ofbinding of VEGF in solution to KDR coated on plates.

cDNAs coding the heavy and light chains of the antibodies were clonedand sequenced. The polypeptides encoded by the cDNAs were aligned andthis alignment is shown in FIG. 2. FIG. 2 shows that two groups ofrelated anti-KDR rabbit monoclonal Abs were obtained. Antibodies 69, 6,71, 43, 81, 4, 30, 54, 57, 50, 68, 56, 83, 36, 77, 95, 14, 42, 27 belongto one group. Antibodies 2, 17, 3, 6, 9 belong to a different group.

FIG. 3 is a multiple sequence alignment of the H3 region of ten rabbitantibody sequences extracted from the Kabat database to illustrate theexpected variation in unrelated antibodies.

Example 3 CDR-Anchored Amplification of Polynucleotides Encoding RelatedAntibodies

Several examples illustrating a method by which the amino acid sequencesof related rabbit antibodies may be obtained by PCR are set forth inFIGS. 4A-4H. In the examples shown in FIGS. 4A-4D, reverse primers thatare complementary to the CDR3 regions of the light chain of antibodies31 (FIG. 4A), 29 (FIG. 4 b), 27 (FIG. 4 c) and 20 (FIG. 4 d) weredesigned and can be used along with a universal forward primer (SEQ IDNO: 118) that binds to a site that is present in all rabbit antibodyheavy chain sequences to amplify coding sequences for relatedantibodies. In the example shown in FIG. 4A, the primers designedagainst sequences that encode antibody 31 are expected to amplify lightchain variable domain sequences for antibodies 11, 12, 2, 25, 22, 27, 3,1, 19, 24, 23, 18, 13, 10 and 21, which are all from the same animal asantibody 31 and are related to antibody 31 by lineage. In the exampleshown in FIG. 4B, the primers designed against sequences that encodeantibody 29 are expected to amplify light chain variable domainsequences for antibodies 8, 9, 16 and 32, which are all from the sameanimal as antibody 29 and are related to antibody 29 by lineage. In theexample shown in FIG. 4C, the primers designed against sequences thatencode antibody 27 are expected to amplify light chain variable domainsequences for other antibodies which are all from the same animal asantibody 27 and are related to antibody 27 by lineage. In the exampleshown in FIG. 4D, the primers designed against sequences that encodeantibody 20 are expected to amplify light chain variable domainsequences for other antibodies which are all from the same animal asantibody 20 and are related to antibody 20 by lineage.

In the examples shown in FIGS. 4E-4H, reverse primers that arecomplementary to the CDR3 regions of the heavy chain of antibodies 31(FIG. 4E), 29 (FIG. 4F), 29 (FIG. 4G) and 21 (FIG. 4H) were designed andcan be used along with a universal forward primer (SEQ ID NO: 148) thatbinds to a site that is present in all rabbit antibody heavy chainsequences to amplify coding sequences for related antibodies. In theexample shown in FIG. 4E, the primers designed against sequences thatencode antibody 31 are expected to amplify heavy chain variable domainsequences for antibodies 2, 17, 22, 25, 12, 1, 24, 19, 25, 11, 31, 3,10, 13, 21, 18 and 23, which are all from the same animal as antibody 31and are related to antibody 31 by lineage. In the example shown in FIG.4F, the primers designed against sequences that encode antibody 29 areexpected to amplify heavy chain variable domain sequences for antibodies8, 9, 16 and 32, which are all from the same animal as antibody 29 andare related to antibody 29 by lineage. In the example shown in FIG. 4G,the primers designed against sequences that encode antibody 27 areexpected to amplify heavy chain variable domain sequences for otherantibodies which are all from the same animal as antibody 27 and arerelated to antibody 27 by lineage. In the example shown in FIG. 4H, theprimers designed against sequences that encode antibody 20 are expectedto amplify heavy chain variable domain sequences for other antibodieswhich are all from the same animal as antibody 20 and are related toantibody 20 by lineage.

What is claimed is:
 1. A method comprising: a) obtaining antibody heavychain sequences and antibody light chain sequences from a population ofB cells of an animal, wherein said population of B cells comprises Bcells that produce antibodies that specifically bind to a targetantigen; b) grouping the antibodies based on their lineage to provide aplurality of groups of antibodies, wherein the antibodies in each grouphave heavy chain CDR3 regions that have 0, 1 or 2 amino acidsubstitutions relative to one another and light chain CDR3 regions thathave 0, 1 or 2 amino acid substitutions relative to one another; c)testing a single antibody from each group of a plurality of the groupsof b) in a first assay to identify a first antibody that has anactivity; and, after said first antibody has been identified: d) testinga further antibody that is in the same group as the first antibody in asecond assay, thereby identifying a second antibody that has saidactivity.
 2. The method of claim 1, wherein step a) comprises obtainingat least 1,000 heavy chain sequences and at least 1,000 light chainsequences from said population of B cells.
 3. The method of claim 1,wherein said population of B cells is enriched by affinity for asubstrate comprising said target antigen.
 4. The method of claim 1,wherein each of said groups of antibodies comprises at least twomembers.
 5. The method of claim 1, wherein said single antibody fromeach of said groups comprises naturally paired heavy chain and lightchain variable domains.
 6. The method of claim 1, wherein said singleantibody from each of said groups comprises non-naturally paired heavychain and light chain variable domains.
 7. The method of claim 1,wherein said further antibodies comprise naturally paired heavy andlight chain variable domains.
 8. The method of claim 1, wherein saidfurther antibodies comprise non-naturally paired heavy and light chainvariable domains.
 9. The method of claim 1, wherein said first andsecond assays are the same.
 10. The method of claim 1, wherein saidfirst and second assays are selected from the group consisting of ablocking assay and a neutralization assay.
 11. The method of claim 1,wherein said animal is immunized with said antigen prior to saidobtaining step a).
 12. The method of claim 1, wherein the animal is arabbit.
 13. The method of claim 1, wherein the antibodies within each ofsaid groups are related to one another by lineage and comprise heavychain CDR3 regions that have 0 amino acid substitutions relative to oneanother and light chain CDR3 regions that have 0 amino acidsubstitutions relative to one another.